BIOTECHNOLOGIES OF USING MULTIPLE EFFECTS OF UGT/IAGLU GENE EXPRESSION FROM MAIZE ZEA MAYS L. TO INCREASE CROP PRODUCTIVITY AND ACTIVE SYNTHESIS OF ANTIGENIC PROTEINS IN THE DEVELOPMENT OF VACCINES AGAINST DANGEROUS VIRAL DISEASES

Abstract

The ugt/iaglu gene was isolated from a sweet corn seedling cDNA library in the laboratory under Professor R.S. Bandurski (MSU, East Lansing, USA) and shared with Dr N. I. Rekoslavskaya for further experimental work. The ugt/iaglu gene encoded the UDPG-transferase, which participated in the metabolism of IAA, the main plant phytohormone, by binding IAA with glucose and thus creating a depot of auxin in plants. In our work, we used the gene ugt / iaglu to improve the hormonal status as much as 20 species of plants both cultivated and wild species by inserting the gene ugt/iaglu in binary agrobacterial vectors. As a result of transformation, an increase of growth, development, rooting, and fruit formation was observed in all transgenic plant species tested. The highest level of free and bound IAA was determined and a very high specific activity of UDPG-transferase in these transgenic plants was found. The transformation characteristics were stable in the 16 seed generations. Due to improvement of hormonal status in transgenic plants, the gene ugt/iaglu was recruited in experience in biotechnological works for developing of preventive vaccines against HIV/AIDS and hepatitis B (HBV) using classical binary agrobacterial vectors. The coat and core proteins of HIV and HBV were barely expressed in transgenic tomato with the low coefficient of tranformation for HIV proteins which was less than 0.001-0.01% and for HBV proteins about 0.01% of yield. But the addition of the ugt/iaglu gene revealed in 50% of surviving explants for HIV tranformants and up to 100% of surviving for HBV transgenic explants. Furthermore, the yields of target vaccine proteins both for HIV and for HBV increased twice or more. We developed our own new plant expression system on the basis of RdRP (RNA dependent RNA polymerase, RNA encoded) from CMV with antisilent RNA 2b encoded in the same frame as RNA 2a. As the second antisilencer gene, we used the ugt/iaglu gene form Z. mays because the ugt / iaglu gene not only stabilized the hormonal homeostasis of tomato transgenic fruits by prolonging its synthesizing activity, but highly increased the accumulation of "early" and "late" proteins of HPV16 high-risk papillomavirus HPV16 during the development of both prophylactic and therapeutic vaccines against cancer. The HPV antigenic protein yield was about 25% per 1 mg of total soluble protein in transgenic tomato fruit. The high yield of antigenic HPV proteins in our plant expression system allowed us to raise antibodies in mice with high titer, strong avidity, and remarkable cross-reactivity with antigens from unrelated HPV families. In our experiments with mice, antibodies to HPV16 L1, HPV18 L1, HPV16 E2, and HPV6 L1 have higher titer and avidity than analogs from the Santa Cruz company. According to the data these multiple enriched functions of the ugt/iaglu gene could not be explained only by the binding and liberation of free auxin during development (for example, fast growth of baby corn after pollination), but by opening new reading frames and accelerating gene expression. Glycosylation and destruction of protein repressors at promoter sites do play important role in this way.